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Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons

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Abstract
Background: Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Until recently, identification of the targeted genes relied on labour intensive and time consuming positional cloning methods. In this study, we outline a straightforward and efficient strategy for fast and comprehensive cloning of amplified and overexpressed genes. Results: As a proof of principle, we analyzed neuroblastoma cell line IMR-32, with at least two amplification sites along the short arm of chromosome 2. In a first step, overexpressed cDNA clones were isolated using a PCR based subtractive cloning method. Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification. Using this approach, amplification of all previously reported amplified genes in this cell line was detected. Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences. Conclusions: The combinatorial strategy of subtractive cDNA cloning and array CGH analysis allows comprehensive amplicon dissection, which opens perspectives for improved identification of hitherto unknown targeted oncogenes in cancer cells.
Keywords
BREAST-CANCER, SOLID TUMORS, PCR, COPY-NUMBER, AMPLIFICATION, MYCN, COAMPLIFICATION, NEUROBLASTOMA-CELL-LINES, COMPARATIVE GENOMIC HYBRIDIZATION, DEAD BOX GENE

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Chicago
De Preter, Katleen, Filip Pattyn, Geert Berx, Kristin Strumane, Björn Menten, Frans Van Roy, Anne De Paepe, Franki Speleman, and Jo Vandesompele. 2004. “Combined Subtractive cDNA Cloning and Array CGH: An Efficient Approach for Identification of Overexpressed Genes in DNA Amplicons.” Bmc Genomics 5.
APA
De Preter, K., Pattyn, F., Berx, G., Strumane, K., Menten, B., Van Roy, F., De Paepe, A., et al. (2004). Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons. BMC GENOMICS, 5.
Vancouver
1.
De Preter K, Pattyn F, Berx G, Strumane K, Menten B, Van Roy F, et al. Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons. BMC GENOMICS. 2004;5.
MLA
De Preter, Katleen, Filip Pattyn, Geert Berx, et al. “Combined Subtractive cDNA Cloning and Array CGH: An Efficient Approach for Identification of Overexpressed Genes in DNA Amplicons.” BMC GENOMICS 5 (2004): n. pag. Print.
@article{209501,
  abstract     = {Background: Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Until recently, identification of the targeted genes relied on labour intensive and time consuming positional cloning methods. In this study, we outline a straightforward and efficient strategy for fast and comprehensive cloning of amplified and overexpressed genes.
Results: As a proof of principle, we analyzed neuroblastoma cell line IMR-32, with at least two amplification sites along the short arm of chromosome 2. In a first step, overexpressed cDNA clones were isolated using a PCR based subtractive cloning method. Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification. Using this approach, amplification of all previously reported amplified genes in this cell line was detected. Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences.
Conclusions: The combinatorial strategy of subtractive cDNA cloning and array CGH analysis allows comprehensive amplicon dissection, which opens perspectives for improved identification of hitherto unknown targeted oncogenes in cancer cells.},
  articleno    = {11},
  author       = {De Preter, Katleen and Pattyn, Filip and Berx, Geert and Strumane, Kristin and Menten, Bj{\"o}rn and Van Roy, Frans and De Paepe, Anne and Speleman, Franki and Vandesompele, Jo},
  issn         = {1471-2164},
  journal      = {BMC GENOMICS},
  keyword      = {BREAST-CANCER,SOLID TUMORS,PCR,COPY-NUMBER,AMPLIFICATION,MYCN,COAMPLIFICATION,NEUROBLASTOMA-CELL-LINES,COMPARATIVE GENOMIC HYBRIDIZATION,DEAD BOX GENE},
  language     = {eng},
  pages        = {14},
  title        = {Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons},
  url          = {http://dx.doi.org/10.1186/1471-2164-5-11},
  volume       = {5},
  year         = {2004},
}

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